The United States is battling an opioid abuse epidemic. More than 2 million individuals are addicted to prescription opioids, and the annual overdose rate from prescription and illicit opioids has quadrupled since 1999. There is an urgent need for safer, less addicting analgesic drugs and for drugs that antagonize opioid- induced adverse effects (e.g., respiratory depression). Mu opioid (mu) G protein coupled receptor (GPCR) activation provides opioid-mediated analgesia. Endomorphin 1 and 2 are endogenous mu-selective opioid tetrapeptide agonists (Tyr-Pro-Trp-Phe-NH2 and Tyr-Pro-Phe-Phe-NH2), and rodent studies suggest these agents provide analgesia without the side effects of motor impairment, respiratory depression, or addiction. Thyrotropin releasing hormone (TRH) GPCR activation provides mild analgesia, hinders opioid tolerance, and stimulates breathing to reverse even deep levels of opioid-induced respiratory depression. Its endogenous agonist, TRH, is a processed tripeptide ((pyro)Glu-His-Pro-NH2). Both endomorphins and TRH peptides, unfortunately, have poor bioavailability due to proteolysis and poor blood brain barrier penetration. Cyclic peptides are a pharmacologic alternative to linear peptides with increased peptidase resistance, increased biologic activity due to rigid structures, and a potential for membrane permeability. In this project, we seek to identify novel cyclic peptide agonist of both the Mu and TRH GPCRs (Aim #1) with therapeutic potential for use as analgesics or breathing stimulant drugs. The new cyclic peptide agonists will be transiently expressed in mammalian cells using modified mRNA (modRNA) (Aim #2) as a novel means of prolonged cyclic peptide drug administration. We will use Split Intein-mediated Circular Ligation of Peptides and Proteins (SICLOPPS), an established system for cyclic peptide production, which relies on a DNA plasmid-encoded self-splicing protein, and which has been used successfully in yeast and mammalian cells. To identify cyclic peptide agonists, we will co-express a library of DNA-encoded cyclic peptides with the mu or TRH GPCRs in an established, engineered his3 auxotrophic yeast strain that responds to surface GPCR activation by growth in histidine-deficient media. We will use modRNA to express cyclic peptides for future in vivo therapeutic delivery. modRNA is mRNA that has been stabilized and made non-immunogenic and non- cytotoxic by substitution of uridine and cytidine with the modified nucleosides pseudouridine and 5- methylcytidine. modRNA provides rapid, robust, titrateable, and time limited in vivo protein production without concern for chromosomal integration in both dividing and non-dividing cells.